Migration of vascular smooth muscle cells (VSMC) plays a key role in both the physiological and the pathophysiological vascular function such as angiogenesis, restenosis and atherosclerosis. Migration of cells in three-dimensional (3D) matrix is a more closely related model system to tissue than conventional two-dimensional (2D) cell migration. However, there is very little research on the mechanisms controlling VSMC migration in 3D matrix. We will use a 3D migration system and VSMC as an in vitro model system for identifying the mechanisms that regulate VSMC migration. It is well established that myosin II is the crucial motor component of muscle contraction and plays a fundamental role in various types of cellular movement. Whereas there are abundant studies of the myosin II motor in VSMC contraction, little is known of the details of how the myosin II motor controls and is regulated in VSMC migration in 3D matrix. In vertebrate, the function of myosin II molecules is dictated by phosphorylation of its regulatory light chain (MLC). Therefore, it has been thought that the phosphorylation of MLC is a key factor in the regulation of numerous cellular movements. In addition to the involvement of MLC phosphorylation in cell motility, non-muscle myosin IIA (NMIIA) and IIB (NMIIB) isoforms have been shown to serve different roles in the regulation of the cell migration in mammalian fibroblasts. Quite recently, we have proposed a new model where myosin II controls cell migration in a negative and positive fashion, which are mediated by cPKC1 and ZIP kinase. Thus, the following Specific Aims will be addressed: Specific Aim 1) To determine the mechanism by which MLC phosphorylation controls the reorganization of the actomyosin cytoskeleton and regulates VSMC migration in the 3D collagen matrix. Analyses will be performed to determine when and where MLC phosphorylation occurs during platelet-derived growth factor (PDGF)-mediated VSMC migration, and to investigate the roles of cPKC1 and ZIP kinase in this process. Specific Aim 2) To determine the roles and mechanisms of non-muscle myosin IIA (NMIIA) and IIB (NMIIB) isoforms in regulating directionality and contractility in VSMC migration in the 3D matrix. RNA interference (RNAi) approaches will be used to examine the roles of different NMII isoforms in VSMC migration in the 3D collagen matrix. Our studies will provide an important foundation of high relevance to motile behavior of VSMC during atherogenesis, which is considered the most critical underlying cause of vascular disease such as heart attack and stroke. Moreover, the studies proposed here will be important for understanding the basic mechanisms by which VSMC migration is achieved in vascular development such as angiogenesis and wound healing. PUBLIC HEALTH RELEVANCE: The proposed project focuses on the regulatory mechanisms of the cellular protein known as myosin II during cell migration. Migration of the vascular smooth muscle cells (VSMC) associated with blood vessels is a fundamental process in the development of atherosclerosis, a major cause of heart attacks and other cardiovascular diseases. Understanding the precise role of myosin II during VSMC migration will help to improve the current understanding of stable blood vessel formation-an essential step toward the treatment of deadly heart disease.